Two adjacent nuclear genes are required for functional complementation of a chloroplast trans-splicing mutant from...

The chloroplast tscA gene from Chlamydomonas reinhardtii encodes a co-factor RNA that is involved in trans-splicing of exons 1 and 2 of the psaA mRNA encoding a core polypeptide of photosystem I. Here we provide molecular and genetic characterization of the trans-splicing mutant TR72, which is defective in the 3'-end processing of the tscA RNA and consequently defective in splicing exons 1 and 2 of the psaA mRNA. Using genomic complementation, two adjacent nuclear genes were identified, Rat1 and Rat2, that are able to restore the photosynthetic growth of mutant TR72. Restoration of the photosynthesis phenotype, however, was successful only with a DNA fragment containing both genes, while separate use of the two genes did not rescue the wild-type phenotype. This was further confirmed by using a set of 10 gene derivatives in complementation tests. The deduced amino acid sequence of Rat1 shows significant sequence homology to the conserved NAD+-binding domain of poly(ADP-ribose) polymerases of eukaryotic organisms. However, mutagenesis of conserved residues in this putative NAD+-binding domain did not reveal any effect on restoration efficiency. Immunodetection analyses with enriched fractions of chloroplast proteins indicated that Rat1 is associated with chloroplast membranes. Using the yeast three-hybrid system, we were able to demonstrate the specific binding of tscA RNA by the Rat1 polypeptide. We propose that the two nuclear factors Rat1 and Rat2 are involved in processing of chloroplast tscA RNA and in subsequent splicing of psaA exons 1 and 2.